PROJECT SUMMARY As a Clinical Instructor in Hematology/Oncology, my career goal is to become an independent physician scientist with a scientific focus on developing new approaches to cancer immunotherapy. This K award period will provide the critical training and support to achieve this goal. This research proposal extends from published work I developed under the mentorship of Dr. Gay Crooks over the past two years. Our interest in the molecular regulation of T cell differentiation from hematopoietic stem and progenitor cells (HSPCs) led us to the discovery that an ?artificial thymic organoid? (ATO) culture system permits in vitro thymic-like differentiation of human HSPCs to fully mature human T cells in a robust and highly reproducible manner. This finding was significant in that prior strategies to direct in vitro T cell differentiation from human HSPCs were largely unable to support the generation of functional T cells. We found that ATOs supported the differentiation of cord blood (CB) HSPCs to CD8+ T cells with a highly diverse T cell receptor (TCR) repertoire comparable to that of nave T cells from the thymus or blood; and further demonstrated that HSPCs engineered with a TCR specific for a tumor-associated antigen efficiently generated mature, tumor-specific T cells in ATOs (Seet et al., Nature Methods, 2017). The focus of this proposal is to exploit a unique property of the ATO system for the de novo discovery of high affinity antigen specific TCRs for cancer immunotherapy. TCR-directed adoptive cell immunotherapy currently relies on the labor-intensive identification from patient samples of TCRs specific to ?public? tumor- associated antigens. However, as many public tumor antigens are non-mutated self-antigens, T cells expressing high affinity TCRs against these targets are typically deleted in the thymus during negative selection, ultimately resulting in the isolation of low affinity TCRs from patient samples. We hypothesize that T cells generated in vitro in ATOs are not subject to negative selection, and thus may present a unique source of high affinity tumor antigen specific TCRs for immunotherapy. In this proposal, I will develop a novel ATO-based platform for the discovery of antigen specific TCRs against public tumor antigens; characterize the nature of ATO-derived TCRs relative to those isolated from peripheral blood; and finally apply these methods to the proof-of-concept capture and preclinical validation of TCRs reactive to prostate cancer specific antigens, a disease in which the identification of tumor-specific TCRs from patients has thus far proved challenging. This is an ambitious project, but one in which the chances of success are maximized through collaboration with the labs of my mentor Dr. Gay Crooks, co-mentor Dr. Owen Witte at UCLA, and Dr. David Baltimore at Caltech. Development of a new technology for rapidly identifying high-affinity TCRs against public tumor antigens would be a significant contribution to the field of immunotherapy research, lead to clinically translatable outcomes, and open promising avenues of further independent research.